Double-stranded nucleic acids containing staggered ends, blunt ends or terminal overhangs of one or two bases can be joined by means of intermolecular or intramolecular ligation reactions. However, ligation of polynucleotides with blunt ends or with one or two base overhangs has been found to be very inefficient. It has been reported that polyethylene glycol (PEG) 6000 can enhance ligation of blunt-ended double-stranded polynucleotides but this reagent interferes with subsequent electroporation of competent cells, a common step in cloning and library preparation.
Using restriction endonuclease-cleaved DNA to create staggered ends, Hayashi et al. (Nucleic Acids Research 14(19):7617-7630 (1986)) ligated double-stranded DNA with a four-base overhang using T4 DNA ligase and 6%-10% (w/v) PEG 6000 to enhance intramolecular ligation and 10% PEG 6000 containing divalent cations or polyamines to enhance intermolecular ligation. Pheiffer et al. (Nucleic Acids Research 11(22):7853-71 (1983)) reported that polymers such as PEG, bovine serum albumen or glycogen could stimulate blunt-end ligation generated by restriction endonuclease cleavage using T4 DNA ligase. In this reference, the authors reported that smaller molecules PEG 200 or PEG 400 had little effect on ligation. Xiao et al. (Molecular Biotechnology 35: 129-133 (2007)) investigated the effects of the organic compounds dimethyl sulfoxide (DMSO), Tween®-20 (Uniqema America LLC, Wilmington, Del.), glycerol, formamide and PEG 6000 on the efficiency and specificity of ligase-detection reactions. They reported that all these compounds except PEG 6000 inhibited the efficiency of ligation although at low concentrations (0-1%) there was a boost in efficiency using Tween-20 which then decreased dramatically. DMSO, glycerol and formamide inhibited the efficiency of the ligation reaction at nick junctions in double-stranded DNA.
Blunt-ended or staggered-ended double-stranded polynucleotides are commonly the product of restriction endonuclease-digestion or fragmentation and polishing. Double-stranded polynucleotides with a single-base overhang are commonly products of PCR amplification using non-proofreading polymerases or the product of adding a dA to a blunt-ended fragment. Alternatively, polishing of the single-base overhang can result in a blunt end. Intermolecular ligation of a restriction endonuclease-cleaved polynucleotide or PCR-amplified polynucleotide to a vector is commonly required prior to a cloning step involving transformation of competent cells.
Intermolecular ligation of single-stranded polynucleotides is undertaken in methods which, for example, involve the ligation of barcode sequences to single-stranded polynucleotides such as cDNA or RNA for cDNA libraries (Edwards et al. Nucleic Acids Research 19(19):5227-32 (1991)) or microRNA cloning (Aravin A, et al. FEBS Lett. 579(26):5830-40 (2005). Epub 2005)) or for sequencing or multi-array techniques. It would be desirable to improve the efficiency of intermolecular and intramolecular ligations of single-stranded and double-stranded polynucleotides without inhibiting downstream reactions.